The aminoacyl-tRNA synthetases (aaRSs) comprise a family of twenty enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the aaRSs. Some aaRSs have developed editing mechanisms to correct misactivated amino acids. These editing aaRSs clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyl-adenylates ("pre-transfer" of arnino acid to tRNA) or misacylated aa- tRNA ("post-transfer"). Although one of these mechanisms may dominate, most aaRSs that edit appear to operate by a mixture of pre-and post-transfer editing, which complicates investigations to determine their respective molecular basis. E. coli leucyl-tRNA synthetase (LeuRS) is unique because it edits exclusively by a post-transfer mechanism. In the past funding cycle, the post-transfer editing activity was abolished and a pre-transfer editing pathway activated in E. coli LeuRS by a limited number of mutations. Thus, the E. coli wild-type and mutant LeuRS provide a powerful model to segregate the two aaRS fidelity mechanisms and characterize molecular determinants that are specific to pre- and/or post-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computational, biochemical, and molecular biology approaches to investigate translocation mechanisms for misactivated aminoacyl- adenylate intermediates in pre-transfer editing and mischarged tRNAs in post-transfer editing. It will also determine the physiological impact of the aaRSs on translational fidelity and cell viability. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs as targets for antibiotic development. It will also enable re-engineering of aaRSs to activate alternate amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or important tools in medicinal and technological applications.